A radioreceptor assay for mass measurement of inositol (1,4,5)-trisphosphate using saponin-permeabilized outdated human platelets.
The binding of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to saponin-permeabilized blood-bank-outdated human platelets, 6 days old, has been characterized (Kd = 3.8 nM; Bmax = 1.7 pmol/mg protein) and used to develop a novel radioreceptor assay which allows the measurement of the Ins(1,4,5)P3 content in resting or agonist-stimulated cells. This assay is as sensitive (0.25 pmol in a 0.25 ml volume), specific, and reproducible as previously proposed methods. In addition, obtaining large batches of the Ins(1,4,5)P3 binding protein by treating outdated platelets with saponin is simple and quick and uses otherwise discarded material. Moreover, the assay is considerably cheaper than commercially available kits. Using this method we confirmed that thrombin evokes a rapid, transient, and dose-dependent increase in the platelet concentration of Ins(1,4,5)P3.
Rivera
J
J
Unit of Hematology and Hemotherapy, School of Medicine, Hospital General Universitario, Murcia, Spain.
Lozano
M L
ML
Gonzalez-Conejero
R
R
Corral
J
J
De Arriba
F
F
Vicente
V
V
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D010539:Permeability; D011869:Radioligand Assay / Q000379:methods* / Q000706:statistics & numerical data; D015203:Reproducibility of Results; D012503:Saponins; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000494:pharmacology
D012503:Saponins; D015544:Inositol 1,4,5-Trisphosphate; D013917:Thrombin